I'm trying to figure out the settings for analysing strand-specific RNA-Seq data from Illumina TruSeq chemistry with rsem-calculate-expression
Specifically, regarding the "--forward-prob" parameter, the manual has this to say:
"....Set to 1 for a strand-specific protocol where all (upstream) reads are derived from the forward strand, 0 for a strand-specific protocol where all (upstream) read are derived from the reverse strand, or 0.5 for a non-strand-specific protocol. (Default: 0.5)"
Question: based upon my understanding of the molecular biology of the TruSeq strand-specific kit, the strand actually getting sequenced in read 1 of a pair is the cDNA sequence, (or in RSEM manual terminology, the reverse strand).
As such, my hunch is that I should be using --forward-prob=0, but I'd like to check with the community to see if I'm getting things right here.
Any help much appreciated,